Detection of Escherichia coli O157:H7 and O104:H4 in patients with diarrhea in Northern Lebanon and characterization of fecal E. coli producing ESBL and carbapenemase genes

Background. While most strains of Escherichia coli (E. coli) are harmless, some are causing intestinal infections of varying severity. The Shiga toxin-producing E. coli (STEC)/ enterohemorrhagic E. coli strains can be associated with fatal clinical manifestations. Of these E. coli Serotypes O157: H7 and O104: H4 were responsible for worldwide epidemics causing thousands of intestinal infections and dozens of deaths. The aim of this research is to investigate the prevalence of E. coli O157: H7 and O104: H4 in the diarrheal stools of 242 Lebanese patients. Materials and methods. This study includes 242 E. coli strains isolated from fecal specimens of patients with diarrhea between February 2013 and May 2014 in the microbiology department of Nini Hospital Laboratory in Tripoli North Lebanon. All specimens were inoculated on sorbitol MacConkey agar. Sorbitol negative strains were investigated for detection of stx1, stx2 and eae genes using real-time PCR. All carbapenem-resistant strains and ESBL producers were investigated by PCR for presence of KPC, IMI, NMC-A, EMS, GHG, VIM, NDM, IMP, OXA-48, blaTEM, blaCTX-M, blaSHV, blaOXA, blaGES and blaPER.. Results. A total of 14 sorbitol negative strains were detected. The search for stx1, stx2 and eae genes showed the presence of a single positive strain for E. coli O157: H7. Out of 242 E.coli strains, 48 (19.8%) were ESBL-positive, 4 (1.6%) were resistant to ertapenem, and all were negative for stx2 genes, The blaCTX-M gene was the most frequent The InTernaTIonal arabIc Journal of anTImIcrobIal agenTs


Introduction
There are about 1.7 billion cases of diarrhea each year worldwide. It was considered the second cause of mortality in children under five years of age and was responsible for 760.000 child deaths per year according the report of WHO in 2013 [1]. Rotavirus and E. coli are the two most common causes of diarrhea in developing countries [1]. The majority of E. coli strains are commensal organisms, however, some were associated with intestinal and extra-intestinal diseases. Among these, the Shiga toxins producing E. coli (STEC)/enterohemorrhagic E. coli (EHEC) strains which are a major cause of bacterial gastroenteritis in the world [2]. There are six pathotypes of intestinal E. coli described in literature, associated with acute diarrheal disease on the basis of their expressed virulence factors; ETEC "enterotoxigenic E. coli," EPEC "enteropathogenic E coli, "EHEC" enterohaemorrhagic E. coli, "EIEC" enteroinvasive E. coli, "EAggEC" enteroaggrega-tive E. coli, and "DAEC" diffusely adhering E. coli [3]. Infection with an EHEC strains can take various clinical forms ranging from asymptomatic carriage to fatal infection. The most common clinical manifestation is Hemorrhagic Colitis (HC) which can be progressed to Hemolytic Uremic Syndrome (HUS), or Thrombotic Thrombocytopenic Purpura (TTP) especially in children and the elderly patients [4]. EHEC O 157 : H 7 is the serotype most important to public health but other serotypes have been frequently involved in sporadic cases and outbreaks [5,6]. During May-June 2011, a large outbreak of E. coli enteroaggregative Hemorrhagic (EAHEC) O 104 : H 4 was reported in Germany and led to HUS in more than 800 patients, many of whom were adults, and ultimately resulted in 54 deaths [6,7]. The severity of this outbreak was significantly higher due to the serotype O 104 : H 4 with over 20% cases of HUS and 1.4% of deaths than with other the serotype O 157 : H 7 . Those HUS cases were affected mainly young children with a mortality rate of 0.5% [8]. While it is recognized that commensal fecal bacteria were important in the emergence of resistance under antibiotic selection pressure. Recently, the main threat is the spread of bacteria with resistant to carbapenems in intestinal of people, since carbapenems are currently the only cure for many serious infections caused by multidrug-resistant (MDR) bacteria [9].
This research aimed to investigate the prevalence of E. coli O 157 and E. coli O 104 in stools of patients with diarrhea in Northern Lebanon by detection their virulence genes stx1, stx2 and eae using real time PCR . Also at the same time to evaluate the rate of fecal E.coli isolates producing ESBL and carbapenems-resistant.

Place and period of the study
This study carried out over the period March 25, 2014 and August 8, 2014 in the microbiology laboratory, health and environment, at the Azm research center for biotechnology which is part of the graduate doctoral school of the Lebanese University.

Fecal E.coli isolates
A total of 242 fecal specimens of diarrheal patients were sent to Microbiology Department of Nini Hospital Laboratory in Tripoli -North Lebanon. All specimens were cultured on sorbitol MacConkey agar (Conda®-Spain); the culture plates were incubated for 24 hours at 37 °C. All E.coli isolates were identified by the Api 20E gallery (Biomérieux®-France) according to the protocol suggested by the manufacturer.

Determination of positive ESBL strains and/or resistant to carbapenems
For the detection of ESBL type resistance and/or the presence of carbapenems resistance, we fol- Finally, the Hodge test was performed for strains resistant to ertapenem [11].

Determination of resistance by production of cephalosporinase.
The inhibition tests are based on the increase in the diameter of inhibition around a disc combining a carbapenem (meropenem or imipenem) and a specific inhibitor of β-lactamases. By testing bacteria producing beta-lactams on a medium containing cloxacillin (250 mg/l) (cephalosporinase inhibitor) and comparatively on a medium without cloxacillin, we can detect a resistance to carbapenems not related to the production of carbapenemase but to the association of cephalosporinase and by default the accumulation of carbapenems which results in a significant increase of the inhibition diameters on the first medium .

Molecular Study of carbapenems resistant strains
A total of 4 E.coli carbapenem-resistance isolates were examined for presence the genes of KPC, IMI, NMC-A, EMS, GHG, VIM, NDM, IMP and OXA-48 ( Table 1). Each isolate is cultivated on nutrient agar (Biorad®, France) and incubated for 24 hours at 37ºC. Few colonies of each isolates were suspended in 0.5 ml of ultrapure sterile water, their DNA were extraction using the kit "GenEluteTM Bacterial Genomic DNA Kit, Sigma-Aldrich.".and stored at -20 °C. The PCR was performed using the following

Discussion
According to the surveillance unit of enteric communicable diseases in Lebanon, a report issued in 2012 showed high occurrence rate of typhoid fever (426cases). The report also observed high incidence of food poisoning (319 cases) and 176 dysentery cases [19].  [32]. In China, the prevalence of carbapenems-resistant among Enterobacteriaceae in the fecal flora of hospitalized patients was 6.6% (20/303 patients), of which 8 isolates were carried bla KPC-2 , bla IMP-4 and bla NDM-1 and typed as carbapenemase producers [33]. Another study conducted in South Korea, showed that the carrier rate of carbapenems-resistant fecal E.coli strains was 1.44% (5/347) [34].
In conclusion, our results shows the importance of investigation continuously the fecal stools of patients for prevalence of MDR E.coli and other enteric bacteria, particularly it is necessary to characterize the ESBL type resistance genes including gene sequencing of carbapenems and perform molecular epidemiological studies by pulsed field gel electrophoresis.
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